Sensitivity of Detection and Variant Typing of SARS-CoV-2 in European Laboratories.

The molecular detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is key for clinical management and surveillance. Funded by the European Centre for Disease Prevention and Control, we conducted an external quality assessment (EQA) on the molecular detection and variant typing of SARS-CoV-2 that included 59 European laboratories in 34 countries. The EQA panel consisted of 12 lyophilized inactivated samples, 10 of which were SARS-CoV-2 variants (Alpha, Beta, Gamma, Delta, Epsilon, Eta, parental B.1 strain) ranging from 2.5 to 290.0 copies/µL or pooled respiratory viruses (adenovirus, enterovirus, influenza virus A, respiratory syncytial virus, or human coronaviruses 229E and OC43). Of all participants, 72.9% identified the presence of SARS-CoV-2 RNA correctly. In samples containing 25.0 or more genome copies/µL, SARS-CoV-2 was detected by 98.3% of the participating laboratories. Laboratories applying commercial tests scored significantly better (P < 0.0001, Kruskal-Wallis test) than those using in-house assays. Both the molecular detection and the typing of the SARS-CoV-2 variants were associated with the RNA concentrations (P < 0.0001, Kruskal-Wallis test). On average, only 5 out of the 10 samples containing different SARS-CoV-2 variants at different concentrations were correctly typed. The identification of SARS-CoV-2 variants was significantly more successful among EQA participants who combined real-time reverse transcription polymerase chain reaction (RT-PCR)-based assays for mutation detection and high-throughput genomic sequencing than among those who used a single methodological approach (P = 0.0345, Kruskal-Wallis test). Our data highlight the high sensitivity of SARS-CoV-2 detection in expert laboratories as well as the importance of continuous assay development and the benefits of combining different methodologies for accurate SARS-CoV-2 variant typing.

Influenza returns with a season dominated by clade 3C.2a1b.2a.2 A(H3N2) viruses, WHO European Region, 2021/22.

In the WHO European Region, COVID-19 non-pharmaceutical interventions continued slowing influenza circulation in the 2021/22 season, with reduced characterisation data. A(H3) predominated and, in some countries, co-circulated with A(H1)pdm09 and B/Victoria viruses. No B/Yamagata virus detections were confirmed. Substantial proportions of characterised circulating virus subtypes or lineages differed antigenically from their respective northern hemisphere vaccine components. Appropriate levels of influenza virus characterisations should be maintained until the season end and in future seasons, when surveillance is adapted to integrate SARS-CoV-2.

Analysis of Diagnostic Findings From the European Mobile Laboratory in Guéckédou, Guinea, March 2014 Through March 2015.

BACKGROUND: A unit of the European Mobile Laboratory (EMLab) consortium was deployed to the Ebola virus disease (EVD) treatment unit in Guéckédou, Guinea, from March 2014 through March 2015. METHODS: The unit diagnosed EVD and malaria, using the RealStar Filovirus Screen reverse transcription-polymerase chain reaction (RT-PCR) kit and a malaria rapid diagnostic test, respectively. RESULTS: The cleaned EMLab database comprised 4719 samples from 2741 cases of suspected EVD from Guinea. EVD was diagnosed in 1231 of 2178 hospitalized patients (57%) and in 281 of 563 who died in the community (50%). Children aged <15 years had the highest proportion of Ebola virus-malaria parasite coinfections. The case-fatality ratio was high in patients aged <5 years (80%) and those aged >74 years (90%) and low in patients aged 10-19 years (40%). On admission, RT-PCR analysis of blood specimens from patients who died in the hospital yielded a lower median cycle threshold (Ct) than analysis of blood specimens from survivors (18.1 vs 23.2). Individuals who died in the community had a median Ct of 21.5 for throat swabs. Multivariate logistic regression on 1047 data sets revealed that low Ct values, ages of <5 and ≥45 years, and, among children aged 5-14 years, malaria parasite coinfection were independent determinants of a poor EVD outcome. CONCLUSIONS: Virus load, age, and malaria parasite coinfection play a role in the outcome of EVD.

Correction: Experimental Treatment with Favipiravir for Ebola Virus Disease (the JIKI Trial): A Historically Controlled, Single-Arm Proof-of-Concept Trial in Guinea.

[This corrects the article DOI: 10.1371/journal.pmed.1001967.].

Experimental Treatment with Favipiravir for Ebola Virus Disease (the JIKI Trial): A Historically Controlled, Single-Arm Proof-of-Concept Trial in Guinea.

BACKGROUND: Ebola virus disease (EVD) is a highly lethal condition for which no specific treatment has proven efficacy. In September 2014, while the Ebola outbreak was at its peak, the World Health Organization released a short list of drugs suitable for EVD research. Favipiravir, an antiviral developed for the treatment of severe influenza, was one of these. In late 2014, the conditions for starting a randomized Ebola trial were not fulfilled for two reasons. One was the perception that, given the high number of patients presenting simultaneously and the very high mortality rate of the disease, it was ethically unacceptable to allocate patients from within the same family or village to receive or not receive an experimental drug, using a randomization process impossible to understand by very sick patients. The other was that, in the context of rumors and distrust of Ebola treatment centers, using a randomized design at the outset might lead even more patients to refuse to seek care. Therefore, we chose to conduct a multicenter non-randomized trial, in which all patients would receive favipiravir along with standardized care. The objectives of the trial were to test the feasibility and acceptability of an emergency trial in the context of a large Ebola outbreak, and to collect data on the safety and effectiveness of favipiravir in reducing mortality and viral load in patients with EVD. The trial was not aimed at directly informing future guidelines on Ebola treatment but at quickly gathering standardized preliminary data to optimize the design of future studies. METHODS AND FINDINGS: Inclusion criteria were positive Ebola virus reverse transcription PCR (RT-PCR) test, age ≥ 1 y, weight ≥ 10 kg, ability to take oral drugs, and informed consent. All participants received oral favipiravir (day 0: 6,000 mg; day 1 to day 9: 2,400 mg/d). Semi-quantitative Ebola virus RT-PCR (results expressed in "cycle threshold" [Ct]) and biochemistry tests were performed at day 0, day 2, day 4, end of symptoms, day 14, and day 30. Frozen samples were shipped to a reference biosafety level 4 laboratory for RNA viral load measurement using a quantitative reference technique (genome copies/milliliter). Outcomes were mortality, viral load evolution, and adverse events. The analysis was stratified by age and Ct value. A "target value" of mortality was defined a priori for each stratum, to guide the interpretation of interim and final analysis. Between 17 December 2014 and 8 April 2015, 126 patients were included, of whom 111 were analyzed (adults and adolescents, ≥13 y, n = 99; young children, ≤6 y, n = 12). Here we present the results obtained in the 99 adults and adolescents. Of these, 55 had a baseline Ct value ≥ 20 (Group A Ct ≥ 20), and 44 had a baseline Ct value < 20 (Group A Ct < 20). Ct values and RNA viral loads were well correlated, with Ct = 20 corresponding to RNA viral load = 7.7 log10 genome copies/ml. Mortality was 20% (95% CI 11.6%-32.4%) in Group A Ct ≥ 20 and 91% (95% CI 78.8%-91.1%) in Group A Ct < 20. Both mortality 95% CIs included the predefined target value (30% and 85%, respectively). Baseline serum creatinine was ≥110 µmol/l in 48% of patients in Group A Ct ≥ 20 (≥300 µmol/l in 14%) and in 90% of patients in Group A Ct < 20 (≥300 µmol/l in 44%). In Group A Ct ≥ 20, 17% of patients with baseline creatinine ≥110 µmol/l died, versus 97% in Group A Ct < 20. In patients who survived, the mean decrease in viral load was 0.33 log10 copies/ml per day of follow-up. RNA viral load values and mortality were not significantly different between adults starting favipiravir within <72 h of symptoms compared to others. Favipiravir was well tolerated. CONCLUSIONS: In the context of an outbreak at its peak, with crowded care centers, randomizing patients to receive either standard care or standard care plus an experimental drug was not felt to be appropriate. We did a non-randomized trial. This trial reaches nuanced conclusions. On the one hand, we do not conclude on the efficacy of the drug, and our conclusions on tolerance, although encouraging, are not as firm as they could have been if we had used randomization. On the other hand, we learned about how to quickly set up and run an Ebola trial, in close relationship with the community and non-governmental organizations; we integrated research into care so that it improved care; and we generated knowledge on EVD that is useful to further research. Our data illustrate the frequency of renal dysfunction and the powerful prognostic value of low Ct values. They suggest that drug trials in EVD should systematically stratify analyses by baseline Ct value, as a surrogate of viral load. They also suggest that favipiravir monotherapy merits further study in patients with medium to high viremia, but not in those with very high viremia. TRIAL REGISTRATION: ClinicalTrials.gov NCT02329054.

Increased immunogenicity of a DNA-launched Venezuelan equine encephalitis virus-based replicon DNA vaccine.

A novel genetic vaccine that is based on a Venezuelan equine encephalitis virus (VEE) replicon launched from plasmid DNA is described. The plasmid encodes a VEE replicon under the transcriptional control of the cytomegalovirus immediate-early promoter (VEE DNA). The VEE DNA consistently expressed 3- to 15-fold more green fluorescent protein in vitro than did a conventional DNA vaccine. Furthermore, transfection with the DNA-launched VEE replicon induced apoptosis and type I interferon production. Inoculation of mice with VEE DNA encoding human immunodeficiency virus type 1 gp160 significantly increased humoral responses by several orders of magnitude compared to an equal dose of a conventional DNA vaccine. These increases were also observed at 10- and 100-fold-lower doses of the VEE DNA. Cellular immune responses measured by gamma interferon and interleukin 2 enzyme-linked immunospot assay were significantly higher in mice immunized with the VEE DNA at decreased doses. The immune responses induced by the VEE DNA-encoded antigen, however, were independent of an intact type I interferon signaling pathway. Moreover, the DNA-launched VEE replicon induced an efficient prime to a VEE replicon particle (VRP) boost, increasing humoral and cellular immunity by at least 1 order of magnitude compared to VEE DNA only. Importantly, immunization with VEE DNA, as opposed to VRP, did not induce any anti-VRP neutralizing antibodies. Increased potency of DNA vaccines and reduced vector immunity may ultimately have an impact on the design of vaccination strategies in humans.

DNA-launched RNA replicon vaccines induce potent anti-Ebolavirus immune responses that can be further improved by a recombinant MVA boost.

There are currently no licensed therapeutic treatment or preventive vaccines against Ebolavirus disease, and the 2013-2016 West African outbreak of Ebolavirus disease spread rapidly and resulted in almost 30,000 cases and more than 11,000 deaths. However, the devastating outbreak has spurred the development of novel Ebolavirus vaccines. Here, we demonstrate that alphavirus-based DNA-launched self-replicating RNA replicon vaccines (DREP) encoding either the glycoprotein (GP) gene or co-expressing the GP and VP40 genes of Sudan or Zaire Ebolavirus are immunogenic in mice inducing both binding and neutralizing antibodies as well as CD8 T cell responses. In addition, antibodies were cross-reactive against another Ebolavirus, although the specificity was higher for the vaccination antigen. DREP vaccines were more immunogenic than recombinant MVA vaccines expressing the same Ebolavirus antigens. However, a DREP prime followed by an MVA boost immunization regimen improved vaccine immunogenicity as compared to DREP and MVA homologous prime-boost immunizations. Moreover, we show that a bivalent approach targeting both Sudan and Zaire Ebolavirus can be employed without significant loss of immunity. This opens for further investigation of a pan-Ebolavirus or even a pan-filovirus vaccine.

Early host CD8 T-cell recovery and sensitized anti-donor interleukin-2-producing and cytotoxic T-cell responses associated with marrow graft rejection following nonmyeloablative allogeneic bone marrow transplantation.

OBJECTIVE: We developed a nonmyeloablative conditioning regimen for allogeneic bone marrow transplantation (BMT) followed by donor lymphocyte infusions (DLI) for treatment of chemotherapy refractory malignancies. Although the majority of patients who receive this regimen achieve lasting mixed or full allogeneic chimerism, approximately 30% show initial mixed chimerism followed by loss of the donor graft. These patients recover host hematopoiesis without significant cytopenias. To assess the role of immunologic rejection in graft loss, we compared T-cell recovery and in vitro alloresponses in six patients who lost their marrow graft to that in 16 concurrent patients with sustained donor chimerism. PATIENTS AND METHODS: Conditioning included pretransplant cyclophosphamide (150-200 mg/kg), thymic irradiation (700 cGy), and pre- and post-transplant equine antithymocyte globulin (ATG; ATGAM). HLA-identical related donor BMT was followed by DLI at approximately day 35 in patients without graft-vs-host disease. RESULTS: The group with transient chimerism showed significantly increased circulating host T-cell (median 416 cells/mm(3) vs 10 cells/mm(3), p<0.05) and CD8 T-cell numbers (354 cells/mm(3) vs 71 cells/mm(3), p<0.05) compared to the group with stable mixed or full donor chimerism within the first 100 days post-BMT. All DLI recipients who lost chimerism following DLI had greater than 80% recipient T cells at the time of DLI, whereas those with persistent chimerism had <60% host T cells. Graft rejection was associated with the development of a sensitized anti-donor bulk cytotoxic T-lymphocyte (CTL) response in 4 of 6 evaluated patients, compared to only 1 of 10 evaluated patients with sustained chimerism (p<0.05). Additionally, 3 of 5 evaluated transient chimeras showed high anti-donor CTL precursor frequencies in limiting dilution assays, and 3 of 4 evaluated transient chimeras showed high anti-donor interleukin-2 (IL-2)-producing T-helper (T(H)) cell frequencies. High anti-donor T(H) or cytotoxic T-lymphocyte precursors were not detected in sustained chimeras. CONCLUSION: These data indicate that loss of chimerism in patients receiving this nonmyeloablative regimen is due to immune-mediated rejection. This rejection appears to bemediated by recovering recipient cytolytic CD8(+) cells as well as IL-2-producing recipient T(H) cells. These data are the first to demonstrate sensitization of recipient anti-donor IL-2-producing cells in association with human marrow allograft rejection.

In-depth analysis of the antibody response of individuals exposed to primary dengue virus infection.

Humans who experience a primary dengue virus (DENV) infection develop antibodies that preferentially neutralize the homologous serotype responsible for infection. Affected individuals also generate cross-reactive antibodies against heterologous DENV serotypes, which are non-neutralizing. Dengue cross-reactive, non-neutralizing antibodies can enhance infection of Fc receptor bearing cells and, potentially, exacerbate disease. The actual binding sites of human antibody on the DENV particle are not well defined. We characterized the specificity and neutralization potency of polyclonal serum antibodies and memory B-cell derived monoclonal antibodies (hMAbs) from 2 individuals exposed to primary DENV infections. Most DENV-specific hMAbs were serotype cross-reactive and weakly neutralizing. Moreover, many hMAbs bound to the viral pre-membrane protein and other sites on the virus that were not preserved when the viral envelope protein was produced as a soluble, recombinant antigen (rE protein). Nonetheless, by modifying the screening procedure to detect rare antibodies that bound to rE, we were able to isolate and map human antibodies that strongly neutralized the homologous serotype of DENV. Our MAbs results indicate that, in these two individuals exposed to primary DENV infections, a small fraction of the total antibody response was responsible for virus neutralization.

Crimean Congo hemorrhagic fever virus infects human monocyte-derived dendritic cells.

For some patients infection with Crimean Congo hemorrhagic fever virus (CCHFV) causes a severe disease characterized by fever, vascular leakage and coagulopathy. Knowledge of CCHF pathogenesis is limited and today there is no information about the specific target cells of CCHFV. In this study we analyzed the permissiveness of human peripheral blood mononuclear cells (PBMCs) including monocyte-derived dendritic cells (moDCs) to CCHFV infection. Interestingly, we found that moDCs are the most permissive to CCHFV infection and this infection induced cytokine release from moDCs. Furthermore, supernatants from infected moDCs were found to activate human endothelial cells.

Dengue virus neutralization by human immune sera: role of envelope protein domain III-reactive antibody.

Dengue viruses (DENV) are the etiological agents of dengue fever (DF) and dengue hemorrhagic fever (DHF). The DENV complex consists of four closely related viruses designated DENV serotypes 1 through 4. Although infection with one serotype induces cross reactive antibody to all 4 serotypes, the long-term protective antibody response is restricted to the serotype responsible for infection. Cross reactive antibodies appear to enhance infection during a second infection with a different serotype. The goal of the present study was to characterize the binding specificity and functional properties of human DENV immune sera. The study focused on domain III of the viral envelope protein (EDIII), as this region has a well characterized epitope that is recognized by strongly neutralizing serotype-specific mouse monoclonal antibodies (Mabs). Our results demonstrate that EDIII-reactive antibodies are present in primary and secondary DENV immune human sera. Human antibodies bound to a serotype specific epitope on EDIII after primary infection and a serotype cross reactive epitope on EDIII after secondary infection. However, EDIII binding antibodies constituted only a small fraction of the total antibody in immune sera binding to DENV. Studies with complete and EDIII antibody depleted human immune sera demonstrated that EDIII binding antibodies play a minor role in DENV neutralization. We propose that human antibodies directed to other epitopes on the virus are primarily responsible for DENV neutralization. Our results have implications for understanding protective immunity following natural DENV infection and for evaluating DENV vaccines.

Comparison of plaque- and flow cytometry-based methods for measuring dengue virus neutralization.

As dengue vaccines enter clinical trials, there is a need for rapid and quantitative assays to measure neutralization. We have developed flow-based neutralization assays which generated results similar to those generated by the established, plaque reduction neutralization test. The flow assays are an improvement, as they use human cells and allow for high-throughput screening.

Inactivation of Hantaan virus-containing samples for subsequent investigations outside biosafety level 3 facilities.

OBJECTIVES: The potential risk of accidental infection by hantaviruses in a clinical or research laboratory necessitates special precautionary measures. A biosafety program must address handling and disposal of infectious materials as well as appropriate virus inactivation or depletion procedures to permit necessary further processing of specimens outside the biosafety level 3 laboratory. METHODS: To study the elimination of hantavirus infectivity, the effects of different chemical and physical inactivation and depletion procedures were investigated on Hantaan virus-containing materials. An infectivity assay for hantaviruses was utilised to verify these procedures which are commonly preceding investigations such as ELISA, flow cytometry analysis, Western blot or immunofluorescence assay. RESULTS: Chemical inactivation with methanol, paraformaldehyde, acetone/methanol and detergent-containing lysis buffer as well as physical forces such as UV irradiation and filtration efficiently reduced viral infectivity in infected cells and their supernatants below the detection limit. CONCLUSION: The virus inactivation and depletion methods described herein are suitable to prepare non-infectious samples for further use in immunological, virological and cell-biological assays.

Shaping phenotype, function, and survival of dendritic cells by cytomegalovirus-encoded IL-10.

Human dendritic cells (DCs) are essential for the antiviral immune response and represent a strategically important target for immune evasion of viruses, including human CMV (HCMV). Recently, HCMV has been discovered to encode a unique IL-10 homologue (cmvIL-10). In this study we investigated the capacity of cmvIL-10 to shape phenotype, function, and survival of DCs. For comparison we included human IL-10 and another IL-10 homologue encoded by EBV, which does not directly target DCs. Interestingly, cmvIL-10 strongly activated STAT3 in immature DCs despite its low sequence identity with human IL-10. For most molecules cmvIL-10 blocked LPS-induced surface up-regulation, confirming its role as an inhibitor of maturation. However, a small number of molecules on LPS-treated DCs including IDO, a proposed tolerogenic molecule, showed a different behavior and were up-regulated in response to cmvIL-10. Intriguingly, the expression of C-type lectin DC-specific ICAM-grabbing nonintegrin, a receptor for HCMV infection found exclusively on DCs, was also enhanced by cmvIL-10. This phenotypic change was mirrored by the efficiency of HCMV infection. Moreover, DCs stimulated with LPS and simultaneously treated with cmvIL-10 retained the function of immature DCs. Finally, cmvIL-10 increased apoptosis associated with DC maturation by blocking up-regulation of the antiapoptotic long form cellular FLIP. Taken together, these findings show potential mechanisms by which cmvIL-10 could assist HCMV to infect DCs and to impair DC function and survival.

Human recombinant neutralizing antibodies against hantaan virus G2 protein.

Old world hantaviruses, causing hemorrhagic fever with renal syndrome (HFRS), still present a public health problem in Asia and Eastern Europe. The majority of cases has been recorded in China. The aim of our study was to generate human recombinant neutralizing antibodies to a hantavirus by phage display technology. To preserve the structural identity of viral protein, the panning procedure was performed on native Hantaan (HTN) (76-118) virus propagated in Vero-E6 cells. In total, five complete human recombinant IgG antibodies were produced in a baculovirus expression system. All of them were able to completely neutralize HTN, and Seoul (SEO) virus in a plaque reduction neutralization test (PRNT). Three of these antibodies could also completely neutralize Dobrava (DOB) virus but not Puumala (PUU) virus. All antibodies bind to Hantaan virus G2 protein localized in the virus envelope. The sequence areas within the HTN (76-118)-G2 protein detected by five selected antibodies were mapped using peptide scans. Two partial epitopes, 916-KVMATIDSF-924 and 954-LVTKDIDFD-963, were recognized, which presumably are of paramount importance for docking of the virus to host cell receptors. A consensus motif 916-KVXATIXSF-924 could be identified by mutational analysis. The neutralizing antibodies to the most widely distributed hantaviruses causing HFRS might be promising candidates for the development of an agent for prevention and treatment of HFRS in patients.

Hantavirus infection of dendritic cells.

Dendritic cells (DCs) play a pivotal role as antigen-presenting cells in the antiviral immune response. Here we show that Hantaan virus (HTNV), which belongs to the Bunyaviridae family (genus Hantavirus) and causes hemorrhagic fever with renal syndrome, productively infects human DCs in vitro. In the course of HTNV infection, DCs did not show any cytopathic effect and viral replication did not induce cell lysis or apoptosis. Furthermore, HTNV did not affect apoptosis-inducing signals that are important for the homeostatic control of mature DCs. In contrast to immunosuppressive viruses, e.g., human cytomegalovirus, HTNV activated immature DCs, resulting in upregulation of major histocompatibility complex (MHC), costimulatory, and adhesion molecules. Intriguingly, strong upregulation of MHC class I molecules and an increased intercellular cell adhesion molecule type 1 expression was also detected on HTNV-infected endothelial cells. In addition, antigen uptake by HTNV-infected DCs was reduced, another characteristic feature of DC maturation. Consistent with these findings, we observed that HTNV-infected DCs stimulated T cells as efficiently as did mature DCs. Finally, infection of DCs with HTNV induced the release of the proinflammatory cytokines tumor necrosis factor alpha and alpha interferon. Taken together, our findings indicate that hantavirus-infected DCs may significantly contribute to hantavirus-associated pathogenesis.

Differential antiviral response of endothelial cells after infection with pathogenic and nonpathogenic hantaviruses.

Hantaviruses represent important human pathogens and can induce hemorrhagic fever with renal syndrome (HFRS), which is characterized by endothelial dysfunction. Both pathogenic and nonpathogenic hantaviruses replicate without causing any apparent cytopathic effect, suggesting that immunopathological mechanisms play an important role in pathogenesis. We compared the antiviral responses triggered by Hantaan virus (HTNV), a pathogenic hantavirus associated with HFRS, and Tula virus (TULV), a rather nonpathogenic hantavirus, in human umbilical vein endothelial cells (HUVECs). Both HTNV- and TULV-infected cells showed increased levels of molecules involved in antigen presentation. However, TULV-infected HUVECs upregulated HLA class I molecules more rapidly. Interestingly, HTNV clearly induced the production of beta interferon (IFN-beta), whereas expression of this cytokine was barely detectable in the supernatant or in extracts from TULV-infected HUVECs. Nevertheless, the upregulation of HLA class I on both TULV- and HTNV-infected cells could be blocked by neutralizing anti-IFN-beta antibodies. Most strikingly, the antiviral MxA protein, which interferes with hantavirus replication, was already induced 16 h after infection with TULV. In contrast, HTNV-infected HUVECs showed no expression of MxA until 48 h postinfection. In accordance with the kinetics of MxA expression, TULV replicated only inefficiently in HUVECs, whereas HTNV-infected cells produced high titers of virus particles that decreased after 48 h postinfection. Both hantavirus species, however, could replicate equally well in Vero E6 cells, which lack an IFN-induced MxA response. Thus, delayed induction of antiviral MxA in endothelial cells after infection with HTNV could allow viral dissemination and contribute to the pathogenesis leading to HFRS.